Báo cáo khoa học: Factor-inhibiting hypoxia-inducible factor (FIH) catalyses the post-translational hydroxylation of histidinyl residues within ankyrin repeat domains pot

Factor-inhibiting hypoxia-inducible factor (FIH) catalyses
the post-translational hydroxylation of histidinyl residues
within ankyrin repeat domains
Ming Yang
1
, Rasheduzzaman Chowdhury
1
, Wei Ge
1
, Refaat B. Hamed
1,2
, Michael A. McDonough
1
,
Timothy D. W. Claridge
1
, Benedikt M. Kessler
3
, Matthew E. Cockman
3,
*, Peter J. Ratcliffe
3,
* and
Christopher J. Schofield
1,
*
1 Chemistry Research Laboratory and Oxford Centre for Integrative Systems Biology, University of Oxford, UK
2 Department of Pharmacognosy, Assiut University, Egypt
3 Henry Wellcome Building for Molecular Physiology, University of Oxford, UK
Introduction
Factor-inhibiting hypoxia-inducible factor (FIH) is an

asparaginyl hydroxylase acting on hypoxia-inducible
factor (HIF), a transcription factor that mediates
the hypoxic response in humans. The FIH-catalysed
hydroxylation of a conserved asparaginyl (Asn) residue
within the
C-terminal transcriptional activation
domain (CAD) of HIF-a reduces the interaction of
HIF with the transcriptional coactivator p300⁄ cAMP-
response-element-binding protein (p300 ⁄ CBP) [1,2],
thereby inhibiting HIF-mediated transcription. The
requirement of molecular oxygen coupled to appropri-
ate kinetic properties for catalysis by the FIH and
Keywords
2-oxoglutarate-dependent dioxygenase;
ankyrin repeat domain; factor inhibiting HIF;
histidinyl hydroxylation; post-translational
hydroxylation
Correspondence
M. E. Cockman, Henry Wellcome Building
for Molecular Physiology, University of
Oxford, Oxford OX3 7BN, UK
Fax: +44 1865 287787
Tel: +44 1865 287785
E-mail:
*These authors contributed equally to this
work
Re-use of this article is permitted in
accordance with the Terms and Conditions
set out at />onlineopen#OnlineOpen_Terms
(Received 10 December 2010, revised 13

January 2011, accepted 18 January 2011)
doi:10.1111/j.1742-4658.2011.08022.x
Factor-inhibiting hypoxia-inducible factor (FIH) is an Fe(II) ⁄ 2-oxogluta-
rate-dependent dioxygenase that acts as a negative regulator of the hypoxia-
inducible factor (HIF) by catalysing b-hydroxylation of an asparaginyl
residue in its C-terminal transcriptional activation domain (CAD). In addi-
tion to the hypoxia-inducible factor C-terminal transcriptional activation
domain (HIF-CAD), FIH also catalyses asparaginyl hydroxylation of many
ankyrin repeat domain-containing proteins, revealing a broad sequence
selectivity. However, there are few reports on the selectivity of FIH for the
hydroxylation of specific residues. Here, we report that histidinyl residues
within the ankyrin repeat domain of tankyrase-2 can be hydroxylated by
FIH. NMR and crystallographic analyses show that the histidinyl hydrox-
ylation occurs at the b-position. The results further expand the scope of
FIH-catalysed hydroxylations.
Database
The coordinates for the structure have been deposited in the Protein Data Bank in Europe
(PDBe; under accession code 2y0i
Structured digital abstract
l
FIH and TNKS-1 hydroxylate by enzymatic study (View Interaction 1, 2)
l
FIH and Tankyrase-2 bind by x-ray crystallography (View interaction)
l
FIH and Tankyrase-2 hydroxylate by enzymatic study (View Interaction 1, 2, 3)
l
FIH and TRPV4 hydroxylate by enzymatic study (View interaction)
l
GABPB2 and FIH hydroxylate by enzymatic study (View interaction)
Abbreviations

AR, ankyrin repeat; ARD, ankyrin repeat domain; CAD, C-terminal transactivation domain of HIF-a; FIH, factor inhibiting HIF; HIF, hypoxia
inducible factor; 2OG, 2-oxoglutarate; siRNA, small interfering RNA.
1086 FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS
HIF-prolyl hydroxylases is proposed to enable them to
act as sensing components for the HIF system [3,4]. In
addition to HIF-a, FIH also catalyses the hydroxyl-
ation of conserved Asn residues within the ubiquitous
ankyrin repeat domain (ARD)-containing protein fam-
ily [5–9]. ARDs are composed of a variable number of
33-residue repeats that individually fold into antiparal-
lel a helices connected by a b hairpin ⁄ loop. The
hydroxylated Asn residues are located within the loop
that links individual ankyrin repeats. Asn hydroxyl-
ation of ARD protein stabilizes the stereotypical ARD
fold [10,11]. Although the physiological function(s) of
Asn hydroxylation of ARDs are unclear, proteomic
and biochemical studies imply that intracellular ARD
hydroxylation on Asn residues may be widespread
[5,8]. Studies on FIH-catalysed ARD hydroxylation
have defined a largely degenerate hydroxylation con-
sensus with very few residues ()8, )1, +10 relative to
the hydroxylation position) showing any substantial
conservation, which is consistent with its ability to
accommodate multiple ARD substrates [12,13]. How-
ever, to date, residues that are actually hydroxylated
by FIH are limited to asparaginyl and in one case,
ankyrinR, an aspartyl residue [14].
Tankyrase is a member of the poly-ADP-ribose
polymerase family, which uses NAD
+

as a cosubstrate
to link ADP-ribose polymers to target proteins, result-
ing in a post-translational modification referred to as
PARsylation [15]. Previous work has identified multi-
ple FIH-dependent Asn hydroxylation sites in the
tankyrase-2 ARD, which were observed to be hydrox-
ylated to differing extents [8]. Here, we report the
unexpected findings that two histidinyl residues in the
tankyrase-2 ARD, located at analogous positions to
the asparaginyl hydroxylation sites, are also substrates
for FIH-catalysed b-hydroxylation. In vitro biochemi-
cal studies suggest that FIH may also catalyse His
hydroxylation in other ARDs. The results expand the
scope of potential 2-oxoglutarate (2OG) oxygenase-
catalysed post-translational modifications.
Results
FIH hydroxylates His 238 and His 553 residues in
tankyrase-2 ARD
Previously, we have reported that various human
ARD-containing proteins undergo hydroxylation at
conserved Asn residues [5,6,8,9]. One of the most highly
modified is tankyrase-2, which undergoes FIH-cataly-
sed hydroxylation at eight Asn residues [8]. Alignment
of the tankyrase-2 ARD revealed two His residues
(His 238 and His 553) embedded within the FIH
hydroxylation consensus comprising an ‘LxxxxxDVH’
motif at positions analogous to proven FIH-catalysed
Asn hydroxylation sites (Fig . 1A) [8]. The positioning
of these His residues within the hydroxylation consen-
sus, coupled with the structural similarity between Asn

and His residues, raised the interesting possibility that
His 238 and ⁄ or His 553 of tankyrase-2 might also be
hydroxylated by FIH. To test this hypothesis, we
prepared synthetic 21-residue peptides encompassing
the two His residues of interest and tested them as FIH
substrates. Significantly, both peptides (TNKS2 223–
243 and TNKS2 538–558) displayed a clear +16 Da
mass increment after reaction with FIH under standard
assay conditions (Fig. 1B). MS ⁄ MS analyses of the
modified TNKS2 538–558 peptide after tryptic digestion
assigned the site of hydroxylation to that corresponding
to His 553 in the tankyrase-2 ARD (Fig. S1).
Having established that His-containing peptides are
FIH substrates in vitro, we then investigated whether
tankyrase-2 might be subject to FIH-catalysed His
hydroxylation in cells. To address this, we transfected
plasmids encoding full-length FLAG-tagged tankyrase-
2 and FIH into 293T cells, immunopurified the mate-
rial by FLAG affinity and subjected it to trypsinolysis
and MS ⁄ MS analysis. Peptides containing both
His residues were observed, and MS ⁄ MS sequencing
assigned hydroxylation at His 238 and His 553
(Fig. 2). As previously observed for hydroxyaspara-
gine-containing peptides [8], under our HPLC condi-
tions, the hydroxyhistidine modification had minimal
effect on the peptide chromatographic properties and
the hydroxylated and nonhydroxylated peptides coelut-
ed (data not shown). The exact masses and retention
times of the His-containing peptides were subsequently
used to assign the hydroxylated and nonhydroxylated

peptides studied by LC ⁄ MS analyses.
To determine whether the His hydroxylation
observed on tankyrase-2 was FIH dependent, we quan-
tified hydroxylation at His 238 and His 553 by LC ⁄ MS
in the presence and absence of small interfering RNA
(siRNA) for FIH. 293T cells were transfected with
siRNA duplexes directed against FIH or a nontargeting
control, then transfected with tankyrase-2 plus empty
vector. As an additional control, and to ensure that
FIH levels were not limiting, tankyrase-2 was cotrans-
fected with FIH. LC ⁄ MS data of one representative
experiment are presented in Fig. 3. Following coexpres-
sion with FIH, the two hydroxylation sites displayed
different levels of hydroxylation; His 238 was hydroxyl-
ated to  30%, whereas His 553 was hydroxylated to
 70%. The preference for His 553 was also observed
under physiological levels of FIH with detectable levels
of hydroxylated peptide ( 15%) observed at His553,
M. Yang et al. FIH-catalysed histidinyl-hydroxylation
FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS 1087
but no appreciable hydroxylation (< 4%) on the
His 238 peptide. Importantly, siRNA-mediated knock-
down of FIH suppressed His 553 hydroxylation to
below the limit of detection, indicating a nonredundant
role for FIH in the catalysis of hydroxyhistidine in the
ARD of tankyrase-2. Consistent with previous work
[8], the relative hydroxylation levels for some previously
identified Asn hydroxylation sites in tankyrase-2
expressed in the presence of endogenous level of FIH
were approximately: Asn 427, 12%; Asn 586, 42%;

Asn 671, 5%; and Asn 739, 60% (tryptic fragments
containing the Asn 203, Asn 271, Asn 518 and Asn 706
hydroxylation sites were not detected, data not shown).
FIH-catalysed histidinyl hydroxylation occurs on
the b-carbon
FIH catalyses the hydroxylation of Asn residues at the
b-position [16]. However, histidine residues are metal
chelators and are prone to oxidation at their imidazole
rings by reactive oxygen species, which are generated
in a controlled manner during the catalytic cycle of
2OG-dependent dioxygenases [17]. Reactive oxygen
species are also proposed to enable self-hydroxylation
of FIH at an active-site tryptophan residue [18]. To
investigate the regiochemistry of the FIH-catalysed
His hydroxylation, we analysed the LC ⁄ MS-purified
TNKS2 538–558 peptide product that had been
hydroxylated by FIH (to  75%) using NMR spec-
troscopy. Compared with the spectrum of the non-
hydroxylated peptide, two new doublet peaks at d
H
4.87 and d
H
5.50 ppm, which are coupled to each other
(J = 3.0 Hz), were observed in the
1
H spectrum of the
hydroxylated TNKS2 538–558 peptide in
2
H
2

O
(Fig. 4). These resonances were assigned to the a- and
b-protons, respectively, of the hydroxylated His 553 in
the TNKS2 538–558 fragment.
1
H–
13
C HSQC analysis
(d
H
a 4.87, d
C
a 57.15 ppm) (d
H
b 5.50, d
C
b 65.15 ppm)
supported this assignment (Fig. S2).
Crystal structure of FIH bound to the tankyrase-2
fragment
To investigate how a His residue is hydroxylated by
FIH, we crystallized FIH in complex with Fe(II),
2OG and the TNKS2 538–558 fragment under anaero-
Fig. 1. Tankryase-2 is a substrate for FIH-
catalysed His hydroxylation. (A) Sequence
alignment of the tankyrase-2 ARD (residues
57–798) [36] demonstrating a 4-repeat
periodicity of the ARD, with insertion
sequences (right) following boxed residues
in the corresponding repeats to the left.

Asn residues that have previously been
identified as FIH substrates [8], and the two
His residues located at the conserved
hydroxylation position, are highlighted in
bold and their residue number shown in
parenthesis on the right. (B) Tankyrase-2
peptides containing His 238 and His 553 are
FIH substrates in vitro. Peptides correspond-
ing to: (I) TNKS2 223–243 (RVKIVQLLLQH-
GADVHAKDKG) and (II) TNKS2 538–558
(RVSVVEYLLQHGADVHAKDKG) were
incubated in the presence of recombinant
FIH and displayed a net +16 Da mass shift
as determined by LC-MS. Subsequent
MS ⁄ MS of the FIH-reacted TNKS2 538–558
peptide assigned the oxidation to His 553
(Fig. S1).
FIH-catalysed histidinyl-hydroxylation M. Yang et al.
1088 FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS
bic conditions [19]. The resultant overall FIH structure
(2.28 A
˚
resolution, Fig. 5A) was similar to reported
FIH structures (rmsd values of 0.30–0.33 A
˚
for Ca
backbone atoms) and reveals that the backbone of
TNKS2 538–558 is bound to FIH in a manner that is
similar overall to analogous ARD fragments that
undergo Asn hydroxylation and a fragment of the

HIF-1a CAD substrate (rmsd for Ca backbone atoms
of TNKS2 versus mNotch-1 and CAD  0.2 A
˚
) [6,19].
At the N-terminus of the bound TNKS2 538–558 frag-
ment, residues 541–546 form a short a-helix, possibly
reflecting that in the ankyrin repeat (AR) of the parent
tankyrase ARD protein (Fig. 5A). At the active site,
the Fe(II) and 2OG are bound as first reported for the
structure of FIH in complex with Fe(II) and a fragment
of the HIF-1aCAD [20]. In the FIHÆTNKS2 538–558
structure, the b-methylene of His 553 is positioned
such that the pro-S hydrogen of its methylene group
projects towards the Fe(II) centre (Fig. 5B), suggesting
that it is hydroxylated to give the 3S-hydroxy product,
as observed for Asn hydroxylation by FIH [16]. Histi-
dine binding at the FIH active site apparently induces
a stacking interaction between the substrate imidazole
and the phenolic rings of Tyr 102
FIH
and His 199
FIH
,
which is one of the iron-complexing residues (Fig. 5B).
Close to the b-methylene of His 553, we observed an
electron density that was refined as a water molecule,
although we cannot rule out the possibility that this
density represents another species (e.g. partial reaction
of the substrate; however, attempted refinements with
hydroxylated His 553 were unsuccessful). The imidaz-

ole side chain of His 553 in the TNKS2 538–558 frag-
ment points towards the c-methylene of the side chain
of Gln 239
FIH
(Fig. 5B). Previous structures have
shown that Gln 239
FIH
binds via hydrogen-bonding
interactions to the side chain of Asn residues that
undergo hydroxylation, for example, Asn 803 of HIF-
1a (Fig. 5C) [6,19]. However, in the TNKS2 538–558
structure, the side-chain amide of Gln 239
FIH
is moved
away from the side chain of the hydroxylated residue
Fig. 2. MS analyses assigning hydroxylation
at His 238 and His 553 in tankyrase-2.
Tankyrase-2 was purified from transiently
transfected 293T cells coexpressing FIH.
(A) MS ⁄ MS spectra of the tryptic peptide
IVQLLLQHGADVHAK derived from
tankyrase-2 (residues 226–240) in the
hydroxylated ([M + 3H]
3+
= m ⁄ z 553.28)
(upper) and nonhydroxylated
([M + H]
3+
= m ⁄ z 548.63) (lower) state.
The hydroxylated species (upper) exhibits

a + 16 Da mass shift on the y-ion series
appearing at y3 and assigning hydroxylation
to His238. (B) MS ⁄ MS of the tankyrase-2
tryptic peptide containing His 553
(VSVVEYLLQHGADVHAK) in hydroxylated
([M + 3H]
3+
= m ⁄ z 627.64) (upper) and
unmodified forms ([M + 3H]
3+
= m ⁄ z
622.30) (lower). For both hydroxylated
spectra, a )2 Da mass shift was commonly
observed on fragment ions containing
hydroxyhistidine, which is consistent with
hydroxylation (+16 Da) followed by dehydra-
tion ()18 Da). Because there was no
evidence for a )2 Da shift on the precursor
ions it is likely that during the collision-
induced dissociation process in the MS ⁄ MS
analyses, the hydroxylated His residue
undergoes dehydration to form the
conjugated a,b-dehydrohistidine product.
Note also there was no evidence for forma-
tion of the dehydrohistidine in the NMR
analyses (Fig. 4).
M. Yang et al. FIH-catalysed histidinyl-hydroxylation
FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS 1089
(i.e. with His 553 compared with a hydroxylated
Asn residue) such that it is positioned to make a hydro-

gen bond with the backbone amide of Tyr 102
FIH
.
Apparently concomitant with this change, the side
chain of Tyr 103
FIH
also moves (Fig. 5B).
Evidence that FIH may catalyse His hydroxylation
in other AR sequences
To investigate whether FIH-catalysed His hydroxyl-
ation can occur in other AR sequences, we searched for
naturally occurring AR sequences containing an
‘LxxxxxDVH’ motif with the His residue located at the
conserved hydroxylation position, and tested the corre-
sponding peptides as FIH substrates (Fig. 6). In addi-
tion to tankyrase-2, peptides derived from tankyrase-1,
GA-binding protein subunit beta-2 (GABPB2) and the
transient receptor potential vanilloid-4 (TRPV4) ARD
all displayed +16 Da mass shifts after reaction with
FIH (Fig. 6B). MS ⁄ MS analyses assigned sites of
hydroxylation to His 711 in the tankyrase-1 sequence
(Fig. S3) and His 265 in the TRPV4 sequence (Fig. S4),
both of which are located within the b-hairpin loop at
the structurally conserved hydroxylation position.
To investigate the relative efficiency of FIH-catalysed
histidinyl versus asparaginyl and aspartyl hydroxyla-
tions under standard conditions, we individually
replaced the His residue at the hydroxylation position
in the TNKS2 538–557 peptide with an Asn (TNKS2_
H553N 538–557) or Asp residue (TNKS2_H553D 538–557)

(Fig. S3). The three tankyrase-2-derived AR peptides
were then incubated with FIH under identical assay
conditions, under which the TNKS2_H553N 538–557
peptide was hydroxylated to near completion (> 95%),
the TNKS2_H553D 538–557 peptide was hydroxylated
to  90%, and the His-containing TNKS2 538–557
peptide was hydroxylated to  60% (Fig. S5). These
observations support the proposals arising from analy-
ses with intact tankyrase-2 protein that FIH-catalysed
hydroxylation of His residues is less efficient compared
with that of Asn or Asp residues, at least within the
same sequence background.
Fig. 3. LC ⁄ MS spectra illustrating the effect of FIH intervention on tankyrase-2 hydroxylation at His 238 and His 553. 293T cells were trea-
ted with siRNAs against FIH (‘FIH siRNA’) or a control sequence (‘Endogenous FIH’ ⁄ ‘FIH overexpression’). After siRNA treatment, cells were
transfected with FLAG–TNKS2 and either pcDNA3 FIH (‘FIH overexpression’) or empty vector (‘FIH siRNA’ ⁄ ‘Endogenous FIH’). FLAG–TNKS2
was immunopurified, digested and analysed by LC ⁄ MS to quantify hydroxylation. The efficacy of the siRNA and plasmid transfections were
confirmed by anti-FIH and anti-FLAG immunoblotting (data not shown). (A) Representative LC ⁄ MS spectra of the 226–240 tryptic fragment
containing His 238. Hydroxylation ( 30%) was observed for His 238 under conditions where the level of FIH was not limiting. (B) Represen-
tative LC ⁄ MS spectra of the 538–555 tryptic fragment containing His 553. Hydroxylation was observed at endogenous level of FIH ( 15%)
and when FIH was overexpressed ( 70%).
FIH-catalysed histidinyl-hydroxylation M. Yang et al.
1090 FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS
Discussion
The results, with both peptide fragments and intact
tankyrase-2 protein in human cells, demonstrate that
FIH can catalyse b-hydroxylation of His residues.
Quantification of the extent of hydroxylation on the
tankyrase-2 protein revealed that His hydroxylations
are not as prevalent as some Asn hydroxylations, rais-
ing the possibility that His hydroxylation is less effi-

cient. Consistent with this, peptide studies comparing
the efficiency of FIH-catalysed hydroxylations on
otherwise identical His-, Asp- and Asn-containing
peptides (based on a tankyrase-2 peptide containing
His 553) indicated a preference for Asn at the target
position (Fig. S5). However, it is important to note
that the efficiency of FIH-catalysed ARD hydroxyl-
ation depends not only on the primary sequence and
the target residue, but also on the position of the AR,
and on the overall fold [8,10,11]. Thus, it is possible
that for other proteins FIH-catalysed His hydroxyl-
ation is more efficient.
We were able to demonstrate that endogenous FIH
levels are sufficient to catalyse His hydroxylation of
ectopically expressed tankyrase-2, but with the avail-
able antibodies we were unable to purify sufficient
endogenous tankyrase-2 for analysis of the modifi-
cation on the native protein. Histidine hydroxylation
was reproducibly observed at one site (His 553) on
transfected tankyrase-2 under physiological levels of
FIH expression, demonstrating site-specific selectivity.
When we have been able to purify and quantitate
endogenous ARD substrates, such as with IjBa [5],
Notch-1 [6] and MYPT-1 [9], and compare the levels
of hydroxylation with their 293T transfected counter-
parts, the data from the two expression systems are in
good agreement. Indeed, we have found that, in these
cases, the level of hydroxylation in protein obtained
from transfected cells tends to under-represent the
Fig. 4. FIH catalysed His-hydroxylation occurs at the b-position. Hydroxylated TNKS2 538–558 peptide (RVSVVEYLLQHGADVHAKDKG) was

produced by incubation with FIH under standard assay conditions (hydroxylated to  75% as assessed by MALDI-TOF analyses), LC-MS
purified and analysed by NMR spectroscopy. (B)
1
H NMR spectrum of the hydroxylated TNKS2 538–558 peptide in
2
H
2
O. The resonances at
4.87 and 5.50 ppm, which are absent in the
1
H NMR spectrum of the nonhydroxylated TNKS2 538–558 peptide (A), are ascribed to the a-
and b-proton, respectively, of the hydroxylated His residues. The resonances for the imidazole ring protons (at positions 2 and 5) are
deshielded in the spectrum of the hydroxylated peptide compared to that of the nonhydroxylated. (C) 2D
1
H–
1
H COSY spectrum of the
hydroxylated TNKS2 538–558 peptide in
2
H
2
O indicating the
1
H–
1
H correlation between resonances arising from the a- and b-hydrogens of
the hydroxylated His residue.
M. Yang et al. FIH-catalysed histidinyl-hydroxylation
FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS 1091
true extent of the observed endogenous modification,

possibly as a result of limiting FIH activity, supporting
the proposed existence of His hydroxylation in cells.
2-OG-dependent dioxygenases catalyse a very wide
range of oxidative reactions, possibly the widest of any
enzyme family [21]. In animals, however, the known
reactions that they catalyse are limited to hydroxyla-
tions and N-methyl demethylations via hydroxylation
of methyl groups. In plants and micro-organisms, they
catalyse a much wider range of reactions, including
hydroxylation and desaturation (e.g. such as occurs in
flavonoid biosynthesis) [22,23]. The observation that
FIH can catalyse histidinyl b-hydroxylation is there-
fore of interest. Together with the recent findings that
FIH can catalyse hydroxylation of an aspartyl residue
in ankyrinR [14], the results presented here suggest
that the substrate selectivity of FIH may be even wider
than previously perceived. The combined results also
raise the possibility that FIH homologues from the
JmjC family of 2OG dioxygenases may have hydroxyl-
ation substrates other than asparaginyl, aspartyl or
lysyl residues.
From a structural perspective, the observations that
FIH has flexibility in the residues that it can oxidize
is interesting because, in terms of sequence selectivity,
FIH is known to be highly promiscuous. The crystal-
lographic analyses suggested how FIH can accommo-
date both amide and imidazole side chains. The
hydroxylated histidine is positioned such that its
b-carbon can undergo hydroxylation, as observed for
asparaginyl substrates of FIH. However, binding of

the imidazole ring of the substrate histidine is differ-
ent to that observed for the amide of asparaginyl
substrates. The nitrogens of the histidinyl imidazole
are not positioned to form hydrogen bonds ( 4A
˚
to
O
e
Gln 239
FIH
). Instead the imidazole ring is sand-
wiched between the side chains of His 199
FIH
that
forms part of the catalytic triad and the aromatic
ring of Tyr 102
FIH
. The side chain of Gln 239
FIH
moves away from the substrate to form a hydrogen
bond with the backbone of Tyr 102
FIH
, with concom-
itant movement of Tyr 103
FIH
(Fig. 5). The difference
in how FIH accommodates Asn and His residues at
the active site likely accounts in part for the fact that
His residues are less efficient substrates than Asn resi-
dues for hydroxylation. Mutagenesis studies on the

importance of individual residues in the HIF-1aCAD
in FIH catalysis have shown that only the hydroxyl-
ated Asn residue is essential [24], and combined
studies on ARD substrates imply that FIH probably
accepts many (perhaps > 100) human ARs as
substrates. Further, FIH accepts both unstructured
substrates, for example, the HIF- a or individual AR
sequences, and structural ARD proteins as substrates.
The promiscuity of FIH is further emphasized by the
work described here.
The physiological significance of FIH-catalysed His
hydroxylation is currently unclear. Taken together, it is
conceivable that His hydroxylation might exert a physi-
ological function either independent of, or in concert
Fig. 5. Structure of the FIH complexes. (A) Surface representation
of the FIHÆTNKS2 538–558 dimer structure (PDB ID: 2Y0I) to 2.28 A
˚
resolution showing apparent electron density for residues Ser 540
to His 553 of the TNKS2 538–558 peptide (2F
o
) F
c
map, contoured
to 1r). (B) Stereoview stick representation of the FIH active site of
the FIHÆTNKS2 538–558 complex (FIH, deep teal; TNKS2 538–558,
yellow; Fe(II), orange). (C) Stereoview stick representation of the
superimposed FIHÆmNotch1 1930–1949 (PDB ID: 3P3N, FIH in
green and N1 1930–1949 in white) and FIHÆHIF-1aCAD 788–826
(PDB ID: 1H2K; FIH in purple and HIF-1aCAD 788-826 in salmon)
complexes. A comparison of all FIH complexes suggests that the

pro-S hydrogen of His 553 in TNKS2 538–558 is likely analo-
gously positioned as that observed for hydroxylated asparagines
in FIHÆmNotch1 1930–1949 (PDB ID: 3P3N) and FIHÆHIF-
1aCAD 788–826 (PDB ID: 1H2K) complexes. (B) and (C) also illus-
trate the differences in side-chain conformation for Gln 239
FIH
and
Tyr 103
FIH
between the FIHÆTNKS2 538–558 and FIHÆmNotch1 1930–
1949 ⁄ FIHÆHIF-1aCAD 788-826 complexes.
FIH-catalysed histidinyl-hydroxylation M. Yang et al.
1092 FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS
with, any of the eight previously assigned hydroxyas-
paragine modifications [8]. It may be that b-hydroxyl-
ation serves to stabilize the ARD fold [10,11] of
tankyrase and, in doing so, modulates the hypoxic
response along with other ARDs by regulating the
amount of FIH that is bound to ARDs and therefore
unavailable for hydroxylation of HIF-a [6,13]. It is also
possible that Asn ⁄ His hydroxylation may modulate
protein–protein interactions of tankyrase, as proposed
for other FIH-catalysed hydroxylations [25]. An attrac-
tive possibility is that His hydroxylation plays a more
direct functional role in redox signalling. Precedent for
this comes from work on the ferric uptake repressor,
PerR in Bacillus subtilis, which is inactivated by
oxidation of the imidazole rings of two Fe(II)-binding
histidinyl residues resulting in suppression of peroxide-
defence genes [17,26]. b-Hydroxyhistidine is also

present in both the bleomycin (Fig. S6) and nikkomycin
antibiotics (Fig. S7), which are biosynthesized by non-
ribosomal peptide synthetases [27–29]. However, the
stereochemistry of the b-hydroxyhistidine derivatives
present in both bleomycin and nikkomycin is 2S,3R-
rather than the likely 2S,3S-stereochemistry of b-hy-
droxyhistidine residues produced by FIH catalysis [16].
Further, in both of these antibiotics, biosynthesis is not
catalysed by 2OG dioxygenases, revealing that nature
has developed more than one route to this unusual
amino acid residue.
Materials and methods
Peptide synthesis
Peptides were prepared using an Intavis Multipep auto-
mated peptide synthesizer with Tentagel-S-RAM resin
(Rapp-Polymere, Tu
¨
bingen, Germany) and a standard
Fig. 6. FIH-catalysed His hydroxylation of
ARDs may be common. (A)
CLUSTALW nongap-
ped multiple sequence alignment of ankyrin
repeat sequences containing a target histi-
dine residue at the conserved hydroxylation
position. Corresponding peptides spanning
the potential histidinyl hydroxylation sites
were tested as FIH substrates in vitro, among
which peptides derived from TNKS1, GAB-
PB2 and TRPV4 demonstrate FIH-dependent
hydroxylation. (B–D) LC ⁄ MS analyses demon-

strating FIH-catalysed His hydroxylation of
the following peptides: (B) TNKS1 381–400;
(C) TNKS1 696–715; and (D) GABPB2 115–135.
(E) MALDI spectra showing the hydroxylation
of the TRPV4 249–269 peptide.
M. Yang et al. FIH-catalysed histidinyl-hydroxylation
FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS 1093
9-fluorenylmethoxycarbonyl ⁄ N,N¢-diisopropylcarbodiimide ⁄
1-hydroxybenzotriazole strategy. Final cleavage using 2.5%
triisopropylsilane in CF
3
COOH yielded the peptides as
C-terminal amides which were precipitated in cold ether,
re-dissolved in 0.1% aqueous CF
3
COOH in water and then
freeze-dried. The masses of the predicted peptide products
were confirmed using a Micromass MALDI-TOF (Waters
Manchester, UK) mass spectrometer.
FIH purification and hydroxylation assays
Recombinant human FIH (with an N-terminal His6 tag)
was produced in Escherichia coli BL21 (DE3) cells and
purified by nickel-affinity chromatography and size-exclu-
sion chromatography as reported previously [2]. In vitro
FIH incubation assays employed 20–60 lm FIH, 100 lm
Fe(II), 100 lm peptide, 1 mm 2OG and 1 mm ascorbate.
The assay mixtures were incubated at 37 °C for 1 h prior
to analysis.
Cell culture and transfection
HEK293T cells were passaged in Dulbecco’s modified

Eagle’s medium supplemented with 10% fetal calf serum
(Sigma, St. Louis, MO, USA), 50 IUÆmL
)1
penicillin,
50 lgÆmL
)1
streptomycin and 2 mml-glutamine. Prior to
plasmid transfection, and where appropriate, the FIH gene
was silenced by delivery of siRNA specific to human FIH
(target F1) or nontargeting dHIF (Drosophila HIF) control.
Cells were transfected twice at 24 h intervals using a 25 nm
dose of duplex and Oligofectamine reagent (Invitrogen,
Carlsbad, CA, USA) in accordance with the manufacturer’s
instructions. Following siRNA delivery, cells were transfect-
ed with FuGENE6 (Roche, Welwyn Garden City, UK) in
dishes of 15 cm diameter using 10 lg of total plasmid DNA
in accordance with the manufacturer’s instructions. Cotrans-
fection of tankyrase-2 (pFLAG ⁄ TNKS2) [8] with FIH
(pCDNA3 ⁄ FIH) [6] or empty vector control (pcDNA3) was
performed at a ratio of 4 : 1 and cells were left for 48 h
before downstream analysis.
Immunoprecipitation and immunoblotting
Cells were lysed in IP buffer (20 mm Tris ⁄ HCl, pH 7.4,
100 mm NaCl, 5 mm MgCl
2
, 0.5% (v ⁄ v) Igepal CA-630)
supplemented with 1 · Complete protease inhibitor cocktail
(Roche) and subject to anti-FLAG immunoprecipitation
using FLAG (M2) affinity gel (Sigma). FLAG-tagged tank-
yrase-2 was eluted in 0.5 m ammonium hydroxide (pH

10.5) and either resolved by SDS ⁄ PAGE and digested
‘in-gel’ or desalted by methanol ⁄ chloroform precipitation
prior to ‘in-solution’ digestion with trypsin as described
previously [8]. To confirm the efficacy of the siRNA-
mediated knockdown and the plasmid co-transfection,
input samples were subject to SDS ⁄ PAGE and immuno-
blotted with antibodies directed against FLAG-epitope
(FLAG M2-HRP; Sigma) or FIH (clone 162c [30]).
Mass spectrometry
Tryptic digest of tankyrase-2 purified from 293T cells were
analysed by nanoUPLC-MS ⁄ MS using a 75 lm inner diam-
eter, 25 cm length C
18
nano-AcquityÔ UPLCÔ column
(1.7 lm particle size; Waters) and a 90 min gradient of
2–45% solvent B (solvent A: 99.9% H
2
O, 0.1% HCOOH;
solvent B: 99.9% MeCN, 0.1% HCOOH) on a Waters
nanoAcquity UPLC system (final flow rate 250 nLÆmin
)1
;
6000–7000 psi) coupled to a Q-TOF Premier tandem mass
spectrometer (Waters). MS analyses were performed in
data-directed analysis mode (MS to MS ⁄ MS switching at
precursor ion counts > 10 and MS ⁄ MS collision energy
dependent on precursor ion mass and charge state). All raw
MS data were processed with Proteinlynx Global Server
software (plgs v. 2.2.5, Waters) including deisotoping. The
mass accuracy of the raw data was calibrated using Glu-

fibrinopeptide (200 fmolÆlL
)1
; 700 nLÆmin
)1
flow rate;
785.8426 Da [M + 2H]
2+
) that was infused into the mass
spectrometer as a lock mass during sample analysis. MS
and MS ⁄ MS data were calibrated at intervals of 30 s.
Assignments of potential hydroxylation sites that were
detected by plgs and mascot were evaluated and verified
by manual inspection. For quantitative comparison of non-
hydroxylated versus hydroxylated peptide peaks, the sum of
all MS spectra containing the relevant precursor ion pairs
are shown and the ratio was calculated by comparing the
sum of ion counts for all isotopic peaks of the correspond-
ing precursor ions. MS ⁄ MS analyses of synthetic peptides
was performed on a SynaptÔ high-definition MS (Micro-
mass Ltd, Manchester, UK) using a 2.1 · 100 mm C
18
Acquity UPLC
Ò
BEH300 column (1.7 lm particle size;
Waters) and a 4 min gradient of 5–50% solvent B (solvent
A: 99.9% H
2
O, 0.1% HCOOH; solvent B: 99.9% MeCN,
0.1% HCOOH) at a flow rate of 0.4 mLÆmin
)1

.LC⁄ MS
was performed at trap CE 6V and transfer CE 4V, and
MS ⁄ MS at trap CE 35V and transfer CE 4V. MALDI-
TOF MS analyses of synthetic peptides were performed on
a Waters MicromassÔ MALDI micro MXÔ mass spec-
trometer in positive ion reflectron mode using a-cyano-4-
hydroxycinnamic acid as the MALDI matrix. Instrument
parameters used were: laser energy, 141%; pulse, 2050 V;
detector, 2700 V; Suppression 1500.
NMR analyses
The TNKS2538–558 peptide (RVSVVEYLLQHGADV-
HAKDKG) was hydroxylated ( 75%) by incubation with
FIH in the presence of 2OG, Fe(II) and ascorbate at 37 °C
for 4 h. The hydroxylation product was purified using a
Vydac 218TP C
18
reversed-phase column pre-equilibrated
in 5% aqueous acetonitrile before running a gradient to
FIH-catalysed histidinyl-hydroxylation M. Yang et al.
1094 FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS
100% acetonitrile over 35 min. Elution was monitored
using a Waters Micromass Quattro micro mass spectro-
meter (in positive ion mode) equipped with a Waters 2777
sample manager and a Waters 1525l Binary HPLC pump
system. Fractions with masses corresponding to anticipated
product were collected (5–10 mL) and lyophilized. The pep-
tide was relyophilized after suspending in 700 lL
2
H
2

O.
For NMR analysis, the sample was dissolved in 75 lLof
2
H
2
O and transferred to a 2 mm NMR tube using a hand
centrifuge. NMR experiments were performed at 310 K
using a Bruker AVIII 700 spectrometer equipped with an
inverse TCI cryoprobe optimized for
1
H observation and
running topspin 2 software. HSQC spectra were collected
using adiabatic 180° CHIRP pulses and TOCSY experi-
ments employed the DIPSI-2 isotropic mixing scheme with
mixing times of 120 ms. Spectra are referenced to the resid-
ual water solvent signal at d
H
4.72 ppm.
Crystallography
Crystals of FIHÆTNKS 538–558ÆFe(II)Æ2OG were obtained
under near anaerobic atmosphere (PO
2
< 0.1 ppm) using
1.6 m ammonium sulphate, 6% (V ⁄ V) PEG400, 0.1 m
Hepes ⁄ Na pH 7.5 [19]. A dataset for a FIHÆFe(II) 2OGÆ
TNKS 538–558 crystal was collected at the Diamond beam-
line I04 with an ADSC Q315 3 · 3 CCD detector and was
processed with automated data reduction software xia2 [31]
and scala (ccp4 suite) [32]. Structure was solved by molec-
ular replacement using phaser (search model PDB ID

1H2K) and was refined with CNS [33]. Iterative cycles of
model building in coot [34] and slowcool-simulated anneal-
ing refinement using the maximum-likelihood function and
bulk-solvent modelling in CNS proceeded until the decreas-
ing R ⁄ R
free
no longer converged. procheck [35] was used
to monitor the geometric quality of the model between
refinement cycles and identify poorly modelled areas need-
ing attention. For data collection and refinement statistics
see Table S1.
Acknowledgements
We are grateful to Dr N-W Chi (University of Califor-
nia, San Diego, CA, USA) for providing the Tankyr-
ase-2 expression construct (pFLAG ⁄ TNKS2). This
work was funded by the Biotechnology and Biological
Sciences Research Council, the European Union and
the Wellcome Trust. RBH is on leave from the Faculty
of Pharmacy, Assiut University, Egypt.
References
1 Lando D, Peet DJ, Gorman JJ, Whelan DA, Whitelaw
ML & Bruick RK (2002) FIH-1 is an asparaginyl
hydroxylase enzyme that regulates the transcriptional
activity of hypoxia-inducible factor. Genes Dev 16,
1466–1471.
2 Hewitson KS, McNeill LA, Riordan MV, Tian YM,
Bullock AN, Welford RW, Elkins JM, Oldham NJ,
Bhattacharya S, Gleadle JM et al. (2002) Hypoxia-
inducible factor (HIF) asparagine hydroxylase is identi-
cal to factor inhibiting HIF (FIH) and is related to

the cupin structural family. J Biol Chem 277,
26351–26355.
3 Flashman E, Hoffart LM, Hamed RB, Bollinger JM Jr,
Krebs C & Schofield CJ (2010) Evidence for the slow
reaction of hypoxia-inducible factor prolyl hydroxy-
lase 2 with oxygen. FEBS J 277, 4089–4099.
4 Koivunen P, Hirsila M, Gunzler V, Kivirikko KI &
Myllyharju J (2004) Catalytic properties of the asparagi-
nyl hydroxylase (FIH) in the oxygen sensing pathway
are distinct from those of its prolyl 4-hydroxylases.
J Biol Chem 279, 9899–9904.
5 Cockman ME, Lancaster DE, Stolze IP, Hewitson KS,
McDonough MA, Coleman ML, Coles CH, Yu X, Hay
RT, Ley SC et al. (2006) Posttranslational hydroxyl-
ation of ankyrin repeats in IkappaB proteins by the
hypoxia-inducible factor (HIF) asparaginyl hydroxylase,
factor inhibiting HIF (FIH). Proc Natl Acad Sci USA
103, 14767–14772.
6 Coleman ML, McDonough MA, Hewitson KS, Coles C,
Mecinovic J, Edelmann M, Cook KM, Cockman ME,
Lancaster DE, Kessler BM et al. (2007) Asparaginyl
hydroxylation of the Notch ankyrin repeat domain by
factor inhibiting hypoxia-inducible factor. J Biol Chem
282, 24027–24038.
7 Ferguson JE III, Wu Y, Smith K, Charles P, Powers K,
Wang H & Patterson C (2007) ASB4 is a hydroxylation
substrate of FIH and promotes vascular differentiation
via an oxygen-dependent mechanism. Mol Cell Biol 27,
6407–6419.
8 Cockman ME, Webb JD, Kramer HB, Kessler BM &

Ratcliffe PJ (2009) Proteomics-based identification of
novel factor inhibiting hypoxia-inducible factor (FIH)
substrates indicates widespread asparaginyl hydroxyl-
ation of ankyrin repeat domain-containing proteins.
Mol Cell Proteomics 8, 535–546.
9 Webb JD, Muranyi A, Pugh CW, Ratcliffe PJ &
Coleman ML (2009) MYPT1, the targeting subunit of
smooth-muscle myosin phosphatase, is a substrate for the
asparaginyl hydroxylase factor inhibiting hypoxia-induc-
ible factor (FIH). Biochem J 420, 327–333.
10 Kelly L, McDonough MA, Coleman ML, Ratcliffe PJ
& Schofield CJ (2009) Asparagine beta-hydroxylation
stabilizes the ankyrin repeat domain fold. Mol Biosyst
5, 52–58.
11 Hardy AP, Prokes I, Kelly L, Campbell ID & Schofield
CJ (2009) Asparaginyl beta-hydroxylation of proteins
containing ankyrin repeat domains influences their sta-
bility and function. J Mol Biol 392 , 994–1006.
M. Yang et al. FIH-catalysed histidinyl-hydroxylation
FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS 1095
12 Cockman ME, Webb JD & Ratcliffe PJ (2009) FIH-
dependent asparaginyl hydroxylation of ankyrin repeat
domain-containing proteins. Ann NY Acad Sci 1177,
9–18.
13 Schmierer B, Novak B & Schofield CJ (2010)
Hypoxia-dependent sequestration of an oxygen sensor
by a widespread structural motif can shape the hyp-
oxic response – a predictive kinetic model. BMC Syst
Biol 4, 139.
14 Yang M, Ge W, Chowdhury R, Claridge TDW,

Kramer HB, Schmierer B, McDonough MA, Gong L,
Kessler BM, Ratcliffe PJ et al. (2010) Asparaginyl and
aspartyl hydroxylation of the cytoskeletal ankyrin
family is catalysed by factor inhibiting hypoxia-induc-
ible factor (FIH). J Biol Chem, doi:10.1074/jbc.M110.
193540.
15 Hsiao SJ & Smith S (2008) Tankyrase function at
telomeres, spindle poles, and beyond. Biochimie 90,
83–92.
16 McNeill LA, Hewitson KS, Claridge TD, Seibel JF,
Horsfall LE & Schofield CJ (2002) Hypoxia-inducible
factor asparaginyl hydroxylase (FIH-1) catalyses
hydroxylation at the beta-carbon of asparagine-803.
Biochem J 367, 571–575.
17 Traore DA, El Ghazouani A, Jacquamet L, Borel F,
Ferrer JL, Lascoux D, Ravanat JL, Jaquinod M,
Blondin G, Caux-Thang C et al. (2009) Structural and
functional characterization of 2-oxo-histidine in
oxidized PerR protein. Nat Chem Biol 5, 53–59.
18 Chen YH, Comeaux LM, Eyles SJ & Knapp MJ (2008)
Auto-hydroxylation of FIH-1: an Fe(II), alpha-ketoglu-
tarate-dependent human hypoxia sensor. Chem Commun
(Camb) 39, 4768–4770.
19 Elkins JM, Hewitson KS, McNeill LA, Seibel JF,
Schlemminger I, Pugh CW, Ratcliffe PJ & Schofield CJ
(2003) Structure of factor-inhibiting hypoxia-inducible
factor (HIF) reveals mechanism of oxidative modifica-
tion of HIF-1a. J Biol Chem 278, 1802–1806.
20 Elkins JM, Ryle MJ, Clifton IJ, Dunning Hotopp JC,
Lloyd JS, Burzlaff NI, Baldwin JE, Hausinger RP &

Roach PL (2002) X-Ray crystal structure of Escherichia
coli taurine ⁄ alpha-ketoglutarate dioxygenase com-
plexed to ferrous iron and substrates. Biochemistry 41,
5185–5192.
21 Flashman E & Schofield CJ (2007) The most
versatile of all reactive intermediates? Nat Chem Biol 3,
86–87.
22 Turnbull JJ, Prescott AG, Schofield CJ & Wilmouth
RC (2001) Purification, crystallization and preliminary
X-ray diffraction of anthocyanidin synthase from
Arabidopsis thaliana. Acta Crystallogr D 57, 425–427.
23 Martens S, Preuss A & Matern U (2010) Multifunctional
flavonoid dioxygenases: flavonol and anthocyanin bio-
synthesis in Arabidopsis thaliana L. Phytochemistry 71,
1040–1049.
24 Wilkins SE, Hyvarinen J, Chicher J, Gorman JJ, Peet DJ,
Bilton RL & Koivunen P (2009) Differences in hydroxyl-
ation and binding of Notch and HIF-1alpha demon-
strate substrate selectivity for factor inhibiting HIF-1
(FIH-1). Int J Biochem Cell Biol 41, 1563–1571.
25 Zheng X, Linke S, Dias JM, Gradin K, Wallis TP,
Hamilton BR, Gustafsson M, Ruas JL, Wilkins S,
Bilton RL et al. (2008) Interaction with factor inhibiting
HIF-1 defines an additional mode of cross-coupling
between the Notch and hypoxia signaling pathways.
Proc Natl Acad Sci USA 105, 3368–3373.
26 Lee JW & Helmann JD (2006) The PerR transcription
factor senses H
2
O

2
by metal-catalysed histidine oxida-
tion. Nature 440, 363–367.
27 Du L, Sanchez C, Chen M, Edwards DJ & Shen B
(2000) The biosynthetic gene cluster for the antitumor
drug bleomycin from Streptomyces verticillus
ATCC15003 supporting functional interactions between
nonribosomal peptide synthetases and a polyketide syn-
thase. Chem Biol 7, 623–642.
28 Calcutt MJ & Schmidt FJ (1994) Gene organization in
the bleomycin-resistance region of the producer organ-
ism Streptomyces verticillus. Gene 151, 17–21.
29 Chen H, Hubbard BK, O’Connor SE & Walsh CT
(2002) Formation of beta-hydroxy histidine in the
biosynthesis of nikkomycin antibiotics. Chem Biol 9,
103–112.
30 Stolze IP, Tian YM, Appelhoff RJ, Turley H,
Wykoff CC, Gleadle JM & Ratcliffe PJ (2004) Genetic
analysis of the role of the asparaginyl hydroxylase
factor inhibiting hypoxia-inducible factor (HIF) in
regulating HIF transcriptional target genes. J Biol Chem
279, 42719–42725.
31 Winter G (2010) xia2: an expert system for macromo-
lecular crystallography data reduction. J Appl Crystal-
logr 43, 186–190.
32 Bailey S (1994) The CCP4 suite: programs for protein
crystallography. Acta Crystallogr D 50, 760–763.
33 Brunger AT, Adams PD, Clore GM, DeLano WL,
Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J,
Nilges M, Pannu NS et al. (1998) Crystallography &

NMR system: a new software suite for macromolecular
structure determination. Acta Crystallogr D 54, 905–
921.
34 Emsley P & Cowtan K (2004) Coot: model-building
tools for molecular graphics. Acta Crystallogr D 60,
2126–2132.
35 Laskowski RA, MacArthur MW, Moss DS &
Thornton JM (1993) PROCHECK: a program to check
the stereochemical quality of protein structures. J Appl
Crystallogr 26, 283–291.
36 Sbodio JI, Lodish HF & Chi NW (2002) Tankyrase-2
oligomerizes with tankyrase-1 and binds to both TRF1
(telomere-repeat-binding factor 1) and IRAP (insulin-
responsive aminopeptidase). Biochem J 361, 451–459.
FIH-catalysed histidinyl-hydroxylation M. Yang et al.
1096 FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS
Supporting information
The following supplementary material is available:
Fig. S1. His 553 in TNK2 538–558 (RVSVVEYLLQH-
GADVHAKDKG) is hydroxylated by FIH.
Fig. S2. 1D
1
H NMR and 2D
1
H–
13
C HSQC analyses
of hydroxylation of the TNKS2 538–558 peptide.
Fig. S3. His 711 in TNKS1 696–715 (RVSVVEYLL-
HHGADVHAKDK) is hydroxylated by FIH.

Fig. S4. His 245 in TRPV4 249–269 (RCKHYVELL-
VAQGADVHAQAR) is hydroxylated by FIH.
Fig. S5. FIH-catalysed His-hydroxylation is less effi-
cient than that of Asn and Asp hydroxylations.
Fig. S6. The presence of b-hydroxyhistidine in the gly-
copeptide antibiotic bleomycin.
Fig. S7. Biosynthesis of b-hydroxyhistidine found in
nikkomycin antibiotics.
Table S1. Refinement statistics for the FIHÆTNKS
538-558ÆFe(II)Æ2OG crystal structure.
This supplementary material can be found in the
online version of this article.
Please note: As a service to our authors and readers,
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should be addressed to the authors.
M. Yang et al. FIH-catalysed histidinyl-hydroxylation
FEBS Journal 278 (2011) 1086–1097 ª 2011 The Authors Journal compilation ª 2011 FEBS 1097